DNA replication, repair and recombination 2 (lecture 4) Flashcards
need to repair damaged DNA, not only from replication errors but ___ that occur in the genome
accidental lesions
Fewer than _____ accidental base changes result in a permanent mutation
1/1000
5000 purine bases are lost every day due to a spontaneous reaction called ______
depurination
Spontaneous ______ of C to U occurs at 100 bases/day
Deamination
Depurination is a result of hydrolysis of the _____ linkage
N-glycosyl linkage (this is the bond that attaches the base to the sugar)
DNA damage can occur from exposure to reactive forms of ___ in the cell or chemicals in environment
O2
UV radiation from sun can produce a covalent linkage between two adjacent pyrimidines (T-T or C-T) this is known as
pyrimidine dimers
if unprepared when DNA is replicated DNA damage can lead to with a ___ or a _____ in the daughter strand
deletion or base pair substitution
what are the 4 types of DNA repair
- Base excision repair
- Nucleotide excision repair
- Transcription-coupled repair
- Double-Strand break repair
What ar the two types of double-stranded break repairs
- Non-homologous end joining
- homologous recombination
There are atleast ____ different types of DNA glycosylases
6
each of the 6 different types of DNA glycosylases recognizes a specific type of ____ and catalyzes it ____
altered base, removal
what type of repair is enzyme mediated “flipping out” of base from the helix
Base excision repair
Explain Base excision repair
-DNA glycosylases travel along DNA using base-flipping to evaluate the status of each base. once the enzyme finds the damaged base that it recognizes, it removes that base from its sugar. The “missing tooth” created by DNA glycosylase and the enzymes AP endonuclease and phosphodiesterase cut the phosphodiester backbone. DNA polymerase adds new nucleotides and DNA ligase seals the nick
once DNA glycosylase finds an incorrect base it cleaves the ___ bond connecting the base with sugar
glycosyl
______ are directly repaired beginning with Ap endonuclease
Depurinations
How does nucleotide excision repair differ from base excision repair
in how damage is removed
Nucleotide excision repair can repair any ____ lesion like those chemically-induced and various pyrimidine dimers
bulky
explain Nucleotide excision pathway
a large multi enzyme complex scans the DNA for a distortion in the double helix, rather than for a specific base change. once it finds a lesion, Excision nuclease cleaves the phosphodiester backbone of the abnormal strand on both sides of the distortion, and a DNA helicase peels away the single-stranded oligonucleotide containing the lesion. the large gap produced in the DNA helix is then repaired by DNA polymerase and DNA ligase
What did Tomas Lindahl win the 2015 nobel prize in chemistry for
- he identified the proteins involved in the base excision repair system
- he identified the first glycosylase
What did Aziz Sancar win the 2015 nobel prize in chemistry for
- identified the proteins involved in nucleotide excision repair system
- identified an enzyme he called photolyase
Cells can preferentially direct DNA repair to sequences that are being actively transcribed by linking ____ with DNA repair
RNA polymerase
The importance of of transcription-coupled excision repair is seen in people with ______ syndrome, which is caused by a defect in this coupling. These individuals suffer from growth retardation, skeletal abnormalities, progressive neural retardation, and severe sensitivity to sunlight. RNA polymerase molecules are permanently stalled at sites of DNA damage
Cockayne
RNA polymerase stalls at lesions and directs
repair machinery there
_______ works with BER, NER and others to repair genes that are being expressed when the damage occurs
Transcription-coupled repair
Transcription-coupled repair is specific for the
strand being transcribed (the non-transcribed strand is repaired at the same rate as DNA not being transcribed)
Cockayne’s syndrome
- Defect in transcription-coupled repair
- Growth retardation, Skeletal abnormalities, and sensitivity to sunlight
- RNA polymerase is permanently stalled at sites of damage in important genes
______ is the simplest purine base capable of pairing specifically with Cytosine
hypoxanthine
Hypoxanthine is the direct deamination product of
adenosine
is the DNA molecule optimally constructed for repair? explain
- Yes
- 2 strands means there is a backup copy
- Nature of 4 buses makes distinction between damaged/undamaged obvious
- Every deamination event forms unnatural base
- Possible reason why RNA is not the hereditary information. (cannot distinguish between deaminated Cytosine and natural uracil)
Deamination of guanine yields
Xanthine
Deamination of cytosine yields
Uracil
Deamination of thymine yields
there is no deamination of thymine because it does not have the amine side group
What is a possible reason why RNA is not the hereditary information
- RNA cannot distinguish between deaminated Cytosine and natural Uracil
Special problem exists with methylated ______ in vertebrate DNA
cytosines
methylated cytosines occurs at some ____ sequences and associated with ______
CpG sequences, inactive genes
Deamination of methyl-Cytosine produces ___ mismatch with ____
natural T mismatched with G
about 3% of the C nucleotides in vertebrate DNAs are methylated to help in
controlling gene expression
only about 3% of cytosine nucleotides in the human genome are methylated but they account for ____ of all point mutations associated with inherited human diseases
1/3
A special DNA glycosylase recognizes and removes the Thymine that is made when methylated cytosines are deaminated (note that this thymine is thus mismatched with G) but the repair is relatively
ineffective
_____ are used when the cell has sustained extensive damage
Translesion Polymerases
less accurate, backup polymerases to repair damage
Translesion polymerases
The highly accurate replicative DNA polymerases stall when they encounter damaged DNA, and in emergencies cells employ versatile, but less accurate, backup polymerases, known as _______, to replicate through the DNA damage
translesion polymerases
humans have ____ different types of translesion polymerases
7
some types of translesion polymerases recognize a specific type of DNA damage and correctly add the nucleotide recede to restore the initial sequence. Other make only ______, especially when the template base has been extensively damaged
good-guesses
Translesion polymerases are not as accurate as the normal replicative polymerases when they copy a normal DNA sequence. because they lack ______ and they ____
they lack exonucleolytic proofreading activity; in addition many are much less discriminating than the replicative polymerase in choosing which nucleotide to incorporate initially.
Because of Translesion polymerases inaccuracy they only add a couple of nucleotides before
the replicative polymerase reassociates
Explain Translesion polymerase use in repairing DNA
A replicative polymerase stalled at a site of DNA damage is recognized by the cell as needing rescue. Specialized enzymes covalently modify the sliding clamp (typically, it is ubiquitylated) which releases the replicative DNA polymerase and, together with damaged DNA, attracts a translesion polymerase specific to that type of damage. once the damaged DNA is bypassed, the covalent modification of the clamp is removed, the translesion polymerase dissociates, and the replicative polymerase is brought back into play
Causes of double-strand breaks
- Ionizing radiation
- replication errors
- oxidizing agents and other metabolites
if double-stranded breaks are left unrepaired chromosomes will
break into smaller fragments and be lost
What are two types of double strand break repair
- non-homologous end joining
- Homologous recombination
_______ brings broken ends together and rejoins by DNA ligation; one or more nucleotides will be lost
non-homologous end joining
what type of double strand break repair predominates in human somatic cells and is generally okay since so little of genome is protein coding
non-homologous end joining
By the time a human reaches the age of 70, the typical somatic cell contains over _____ such scars, distributed throughout its genome, representing places where DNA has been inaccurately repaired by non homologous end joining
2000
explain non homologous end joining
used to fix double strand breaks (usually takes place when cells have not yet duplicated their DNA)
Double stranded break leads to nuclease processing both the DNA ends (this is because often the nucleotides around the break are damaged or altered) the sites are then rejoined by DNA ligation
_____ ensure the completion of one stage in the cell cycle before the next can begin
Checkpoints
Presence of DNA damage triggers various checkpoints such as
- blocks entry from G1 into S phase
- Slows down progression through S phase
- blocks transition form G2 to M phase
Checkpoints give the cell extra time to
repair DNA damage
a kinase that generates intracellular signals that alert the cell to DNA damage and up regulate expression of DNA repair genes
ATM protein
Mutation in ATM protein lead to
ataxia telengiectasia (AT)
Ataxia telengiectasia (AT)
- caused by mutations in ATM protein
- symptoms include neurodegeneratiion, predisposition to cancer, genome instability due to unrepaired DNA lesions
Genetic exchange between a pair of homologous DNA sequences
Homologous recombination
functions of homologous recombination
- repair of double-standed breaks
- especially at stalled or broken replication forks
- Exchange of genetic information to create new combinations of genetic sequences
- crossing over and gene conversion in meiosis - Mechanical role in assuring accurate chromosome segregation
Homologous recombination is a fundamental process and common to
all organisms
_____ guides homologous recombination
base-pairing
DNA duplexes ___ each other looking for regions of homology
“sample”
DNA double helix reforming from its separated single strands
Hybridization (also called renaturation)
Once a region of homology is found
the single strands rapidly pair up
base-pairing of homologous recombination can create a double helix from strands that originate from different molecules but are complementary to one another (or atleast largely complementary) is called
heteroduplex DNA
Explain repairing double stranded breaks by homologous recombination
- 5’ ends degraded by exonuclease
- one 3’ end invades homologous template and primes repair DNA synthesis
- The newly synthesized 3’ end of the invading strand is then able to anneal to the other original 3’ overhang in the damaged chromosome through complementary base pairing
- gaps are filled in and ligated
DNA hybridization requires a _____
single-stranded DNA (freed from pairing with complement so it can pair with the 2nd strand)
DNA strand exchange is carried out by the _____ protein
RecA/Rad51
The single-stranded invading strand is directed by _______ and other proteins
RecA (bacteria) Rad51 in eukaryotes
RecA/Rad51 binds cooperatively to single stranded DNA and holds it in a unique configuration, with the backbone _____
stretched out
RecA/Rad51 moves the single strand down the duplex searching ____ nucleotides at a time
3
Once RecA/Rad51 has identified a homologous sequence strand invasion occurs, forming a heteroduplex. This invasion requires an extended stretch of how many homologous base pairs
at least 15
_____ can rescue broken DNA replication forks
Homologous Recombination
The first step in repairing a broken replication fork with homologous recombination is after the replication fork breaks the
exonuclease degrades 5’ end
what is the second step in homologous recombination repair of a broken replication fork (caused by a nick in one strand)
Strand invasion (note this is after exonuclease degrade the 5’ end of the broken strand). This pairs single-stranded DNA with complementary strand in different double-stranded helix. Forms a region of heteroduplex DNA
Accurate repair process can still cause problems for the cell . such as
- use of a non-functioning homolog to “repair” the other homolog
- loss of heterozygosity (this is a critical first step in cancer development and rare occurrence)
Processing of broken ends is coordinated with the ____
cell cycle
Nucleases for generating 3’ invading strand are only active in _______ and ____ phase
S and G2 phase
Nucleases for generating 3’ invading strand are only active in S and G2 phase this ensures a ______ or ____ will be the most likely template for repair
replicated chromosome or sister chromatid
Give examples of how the cell is able to prevent DNA repair in the absence of damage
- Loading of RecA on DNA is tightly controlled
- Repair proteins dispersed throughout cell
mutations in proteins involved in recombination can cause
cancer
mutations in ____ and ____ lead to increased rates of breast cancer
Brca1 and Brca2
_____ regulates the processing of broken ends of chromosome
Brca1
____ maintains Rad51 (RecA) inactive until it is at site of damage
Brca2
Brca1 regulates the processing of broken ends of chromosomes. A mutation in this protein leads to
use of non-homologous end-joining processes
Brac2 maintains Rad51 (RecA) inactive until it is safe at site of damage. mutations in this can lead to
- Rad51 (RecA) an become active at times when not desired
what type of double-strand break repair process does not require a template, creates mutation at site of repair, and can also create translocations
non-homologous end joining
What type of double-strand break repair process use daughter strand DNA duplex as template, no loss or alteration of DNA at repair site, Can repair other types of DNA damage, mechanism and proteins conserved in all organisms
Homologous recombination
____ complex recognizes double-stranded breaks and mobilizes additional proteins to repair the damage
Mre11 (this is a nuclease that processes damaged DNA in preparation for homologous recombination)
